Pituitary adenylate cyclase activating polypeptide induces multiple signaling pathways in rat peritoneal mast cells

Pituitary adenylate cyclase activating polypeptide (PACAP) is a high-affinity ligand for at least two types of G-protein coupled receptors, the PACAP type 1 and type 2 receptor. In this study it is demonstrated that the C-terminal PACAP-fragment PACAP(6-27) stimulates serotonin release from rat peritoneal mast cells with higher potency (EC50: 0.2 vs. 2.0 microM) than the PACAP receptor ligand PACAP(1-27). PACAP-induced degranulation of rat peritoneal mast cells was abolished by pertussis toxin and by benzalkonium chloride (IC50: 9.1 microg/ml) indicating the involvement of heterotrimeric G-proteins of the Gi-type. The PACAP effect was also reduced by inhibitors of the phosphatidylinositol specific phospholipase C ((U73122), IC50: 4 microM; (ET-18-O-CH3), IC50: 18 microM), by D609, a specific inhibitor of the phosphatidylcholine specific phospholipase C (IC50: 41 microM), by the protein kinase C-inhibitor staurosporine (IC50: 0.6 microM) and by the lipoxygenase inhibitor nordihydroguaiaretic acid (NGDA) but not by indomethacin. It is concluded that PACAP peptides stimulate secretion in rat peritoneal mast cells in a PACAP receptor-independent manner, probably via direct activation of heterotrimeric G-proteins of the Gi-type; these G-proteins may lead to a sequential activation of different signaling cascades (see above), which may converge at the level of one or more staurosporine-sensitive protein kinase.     

Ontogeny of calretinin expression in rat dorsal root ganglia

The relationship between the expression of calretinin and the maturation level of peripheral sensory neurons was investigated by means of immunohistochemistry and immunoblot analysis. Our immunohistochemical results show that calretinin is expressed during two different developmental phases in rat dorsal root ganglia. The early phase lasts between embryonic days 11 and 14, when calretinin is detectable in the majority (75%) of the cells. A second phase starts at embryonic day 17 and lasts throughout the whole postnatal life, when calretinin is expressed only in a small proportion of the neurons (less than 8%). Between these two periods no calretinin is found in the ganglia. These changes in calretinin expression during embryonic development were confirmed by Western blot analysis. The early expression of calretinin in dorsal root ganglion cells suggests that calretinin may act as a calcium regulator until neurotrophins take over the precise tuning of intracellular calcium concentration.     

Islet amyloid polypeptide and calcitonin gene-related peptide expression are down-regulated in dorsal root ganglia upon sciatic nerve transection

Islet amyloid polypeptide (IAPP) is structurally related to calcitonin gene-related peptide (CGRP) and has been implicated in glucose homeostasis and diabetes pathogenesis because it is expressed in insulin cells and forms amyloid in pancreatic islets from type II diabetic patients. IAPP is also constitutively co-expressed with CGRP in rat sensory neurons. Whether expression of IAPP is altered by nerve injury with or without regeneration was investigated in adult rats subjected to unilateral sciatic axotomy; IAPP and CGRP expression were determined by quantitative in situ hybridization and immunocytochemistry at days 3, 10 and 30 after axotomy. In ipsilateral L4-L5 dorsal root ganglia (DRG), the percentages of nerve cell profiles labelled for IAPP and CGRP mRNA were reduced at all time points studied. IAPP and CGRP mRNA expression were lower in nerve cell profiles in ipsilateral DRGs compared to the contralateral side after axotomy alone whereas epineurial nerve suture maintained or restored IAPP and CGRP expression. The numbers of IAPP- and CGRP-immunoreactive DRG nerve cell profiles and dorsal horn fibers were reduced on the ipsilateral side at all time points. Thus, IAPP and CGRP expression are down-regulated upon axotomy. Nerve repair maintains or restores IAPP and CGRP expression in individual neurons but does not prevent the loss of CGRP/IAPP phenotype of some of these neurons in response to axotomy.     

Gene expression of pituitary adenylate cyclase activating polypeptide (PACAP) in the rat hypothalamus

Pituitary adenylate cyclase activating polypeptide (PACAP) isolated from ovine hypothalamus is considered to be a member of the vasoactive intestinal peptide/glucagon/secretin/growth hormone-releasing hormone family of peptides. Two forms of PACAP, PACAP38 and PACAP27, have been demonstrated in the rat hypothalamus. The PACAP precursor contains another peptide called PACAP-related peptide (PRP), but so far no information on this peptide in tissue exists. We have developed three radioimmunoassays specific for PACAP38, PACAP27 and PRP and demonstrate that all three preproPACAP peptides are expressed in the rat hypothalamus, the PACAP38/PACAP27 ratio being around 60 and the PACAP38/PRP ratio being around 10. HPLC analysis of hypothalamic extract showed that PACAP38 and PACAP27 are found in only one form corresponding to the respective synthetic peptides, whereas PRP eluted in two peaks, the predominant form corresponding to synthetic PRP1-29. The cellular distribution of PACAP38, PACAP27, and PRP and corresponding mRNA in the hypothalamus was determined with immunohistochemistry and in situ hybridization histochemistry. PACAP- and PRP-immunoreactive neuronal perikarya were observed in the medial parvocellular part of the paraventricular nucleus (PVN) in colchicine pretreated rats. Some cell bodies of magnocellular variety were found in the PVN. PACAP mRNA containing cells were observed in moderate numbers in the same parts of the paraventricular nucleus. PACAP- and PRP immunoreactive fibres and varicosities were distributed in the PVN and in the periventricular nucleus. These data show that PACAP38, PACAP27 and PRP are expressed in the parvocellular part of the PVN, implying roles as hypothalamic regulatory peptides.     

Differential time course of neuronal and glial apoptosis in neonatal rat dorsal root ganglia after sciatic nerve axotomy

Sensory neurons in neonatal rat lumbar dorsal root ganglia die after sciatic nerve axotomy, and previous studies have estimated the total cell loss to be 40-95%. We have used the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) technique, combined with immunohistochemistry, to investigate the contribution of apoptosis to the cell loss that occurs after unilaterally transecting the sciatic nerve of new-born rats. TUNEL-positive cells were detected 1 day post-lesion, and their number peaked 3 days after the injury. Combining TUNEL labelling with immunohistochemistry, for neuron-specific neurofilament 150 kDa, or glial-specific S-100beta, enabled us to identify dying neurons and dying glia. One day after axotomy, most of the TUNEL-positive cells (58%) were neurons, whereas 3 days post-injury, only a small number of dying cells (6%) were neuronal. This lower incidence was due to a decrease in neuronal death and an increase in glial death. The glia in the dorsal root ganglia therefore die subsequent to the neurons. The apoptotic nature of the cell death was confirmed by electron microscopy, with fine structural features of apoptotic cell death, e.g. chromatin compaction and membrane blebbing, being observed in both glia and neurons. Our results confirm that extensive apoptosis occurs in the neonatal lumbar dorsal root ganglia after sciatic nerve section, and show that neurons and glial cells die with different time-courses. The results suggest a neuron-glia trophic interdependence in the dorsal root ganglia.     

Pituitary adenylate cyclase activating polypeptide (PACAP) stimulates mitogen-activated protein kinase (MAPK) in cultured rat astrocytes

Astrocytes, a subtype of glial cells, have been demonstrated to have an abundant number of receptors for pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide of the VIP/secretin family which stimulates cAMP accumulation 1000 times more potent than VIP in astrocytes. PACAP is reported to stimulate the proliferation of astrocytes at low concentrations at which it does not yet stimulate the cAMP accumulation. In the present study, we examined the effect of PACAP on the activation of mitogen-activated protein kinase (MAPK), one of the important intracellular signals for the proliferation, and compared it with that of epidermal growth factor (EGF). To investigate the activation of MAPK, we focused on ERK2, one of MAPK, in cultured rat astrocytes. The activation of ERK2 was determined by immunoblotting and measurement of the activity in terms of the phosphorylating activity of immunoprecipitates with MAPK antibody on myelin basic protein. One pM of PACAP38 temporarily activated ERK2 at 10 min. In contrast, EGF activated ERK2 from 10 min to 60 min continuously. As for the dose-response effect, PACAP stimulated ERK2 at as low a concentration as 10-14 M and peaked at 10-12 M. Thereafter, its activating effect gradually decreased at 10-10 M and returned to the basal level at 10-8 M, forming a bell-shaped dose-dependency. Neither an inhibitor of PKA (H89) nor inhibitors of PKC (staurosporine and calphostin C) had any effect on the ERK2 activation induced by 1 pM PACAP38. Dibutyryl cAMP suppressed ERK2 activity in a dose-dependent manner. These data clearly demonstrated that PACAP stimulates MAPK in both a PKA- and a PKC-independent manner in cultured rat astrocytes.     

c-Jun expression in adult rat dorsal root ganglion neurons: differential response after central or peripheral axotomy

The response of the mature central nervous system (CNS) to injury differs significantly from the response of the peripheral nervous system (PNS). Axotomized PNS neurons generally regenerate following injury, while CNS neurons do not. The mechanisms that are responsible for these differences are not completely known, but both intrinsic neuronal and extrinsic environmental influences are likely to contribute to regenerative success or failure. One intrinsic factor that may contribute to successful axonal regeneration is the induction of specific genes in the injured neurons. In the present study, we have evaluated the hypothesis that expression of the immediate early gene c-jun is involved in a successful regenerative response. We have compared c-Jun expression in dorsal root ganglion (DRG) neurons following central or peripheral axotomy. We prepared animals that received either a sciatic nerve (peripheral) lesion or a dorsal rhizotomy in combination with spinal cord hemisection (central lesion). In a third group of animals, several dorsal roots were placed into the hemisection site along with a fetal spinal cord transplant. This intervention has been demonstrated to promote regrowth of severed axons and provides a model to examine DRG neurons during regenerative growth after central lesion. Our results indicated that c-Jun was upregulated substantially in DRG neurons following a peripheral axotomy, but following a central axotomy, only 18% of the neurons expressed c-Jun. Following dorsal rhizotomy and transplantation, however, c-Jun expression was upregulated dramatically; under those experimental conditions, 63% of the DRG neurons were c-Jun-positive. These data indicate that c-Jun expression may be related to successful regenerative growth following both PNS and CNS lesions.     

Expression of neurotrophin mRNAs in the dorsal root ganglion after spinal nerve injury

This study aimed to characterize the interaction between nitric oxide (NO)- and cAMP-related pathways in the control of renal blood flow. Using the isolated perfused rat kidney model, we determined the effects of inhibition of NO formation by Nomega-nitro-L-arginine methyl ester (L-NAME; 1 mmol/L) and of NO administration by sodium nitroprusside (SNP, 10 micromol/L) on renal vascular resistance under conditions of elevated vascular cAMP levels. cAMP levels were increased either by adenylate cyclase activation via isoproterenol or by inhibition of cAMP phosphodiesterases (PDEs) 1, 3, and 4. We found that L-NAME markedly increased vascular resistance and that this effect was completely reversed by SNP. Both isoproterenol and inhibitors of the cAMP PDEs lowered basal vascular resistance. In the presence of isoproterenol (3 nmol/L) and inhibitors of PDE-1 [8-methoxymethyl-l-methyl-3-(2-methylpropyl)-xanthine; 8-MM-IBMX, 20 micromol/L] and PDE-4 (rolipram, 20 micromol/L), L-NAME again substantially increased vascular resistance, and this effect of L-NAME was completely reversed by SNP. In the presence of the PDE-3 inhibitors milrinone (20 micromol/L) and trequinsin (200 nmol/L), however, both L-NAME and SNP failed to exert any additional effects. Because PDE-3 is a cGMP-inhibited cAMP PDE and because the vasodilatory effect of SNP was abrogated by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) (20 micromol/L), our findings are compatible with the idea that an action of NO on PDE-3 could account for the vasodilatory properties of NO on the renal vasculature. Moreover, our findings suggest that PDE-3 activity is an important determinant of renal vascular resistance.     

Hemodynamic actions of systemically injected pituitary adenylate cyclase activating polypeptide-27 in the rat

The aims of this study were (1) to characterize the hemodynamic mechanisms underlying the hypotensive effects of pituitary adenylate cyclase activating polypeptide-27 (PACAP-27 0.1-2.0 nmol/kg, i.v.) in pentobarbital-anesthetized rats, and (2) to determine the roles of the autonomic nervous system, adrenal catecholamines and endothelium-derived nitric oxide (NO) in the expression of PACAP-27-mediated effects on hemodynamic function. PACAP-27 produced dose-dependent decreases in mean arterial blood pressure and hindquarter and mesenteric vascular resistances in saline-treated rats. PACAP-27 also produced pronounced falls in mean arterial blood pressure in rats treated with the ganglion blocker, chlorisondamine (5 mg/kg, i.v.). The hypotensive and vasodilator actions of PACAP-27 were not attenuated by the beta-adrenoceptor antagonist, propranolol (1 mg/kg, i.v.), or the NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME 50 micromol/kg, i.v.). PACAP-27 produced dose-dependent increases in heart rate whereas the hypotensive response produced by the nitrovasodilator, sodium nitroprusside (10 microg/kg, i.v.), was associated with a minimal tachycardia. The PACAP-27-induced tachycardia was unaffected by chlorisondamine, but was virtually abolished by propranolol. These results suggest that the vasodilator effects of PACAP-27 are due to actions in the microcirculation rather than to the release of adrenal catecholamines and that this vasodilation may not involve the release of endothelium-derived NO. These results also suggest that PACAP-27 produces tachycardia by directly releasing norepinephrine from cardiac sympathetic nerve terminals rather than by direct or baroreceptor reflex-mediated increases in sympathetic nerve activity.     

The time-course of abeta-evoked c-fos expression in neurons of the dorsal horn and gracile nucleus after peripheral nerve injury

We have examined the mechanisms underlying Abeta-evoked c-fos expression in the dorsal horn and gracile nucleus following either sciatic nerve section or crush injury. The results indicate that in the spinal cord Abeta-evoked c-fos does not depend on primary afferent sprouting but is associated with the disconnection from the peripheral target since its expression in the dorsal horn reverts to normal after crush injury when regeneration occurs but persists after cut and ligation where regeneration is prevented. In contrast, however, Abeta-evoked c-fos expression in the gracile nucleus may be under some other control since its expression appears independent of peripheral nerve regeneration.     

Increased spike-frequency adaptation and tea sensitivity in dorsal root fibers after sciatic nerve injury

Excitability of rat dorsal root axons were studied 3 weeks after injury to the sciatic nerve. Whole nerve recordings were obtained from injured and control nerves in a sucrose gap chamber. Constant current depolarization pulses (30-200 ms) applied approximately 50% above the stimulus strength required for maximal amplitude compound action potentials (CAPs) evoked a burst of action potentials in the dorsal root which displayed spike adaptation. The depolarization-induced burst response of the dorsal roots was greatly reduced after crush or transection of the sciatic nerve. However, application of the potassium channel blocker, tetraethylammonium (TEA), restored the burst discharge in injured dorsal root axons. Brief tetanic stimulation of the dorsal root also induced an afterhyperpolarization (AHP) that was twice as large in the transection group as compared to the control group, and which was blocked by TEA. There were no changes seen in the amplitude of the compound action potential, frequency-following characteristics, refractory properties, or 4-AP sensitivity in the dorsal roots after peripheral nerve injury. These results suggest that there is enhanced spike adaptation that occurs at the same time as an increase in the sensitivity to the potassium channel blocker, TEA, in axon regions proximal to the site of nerve injury and have implications for the pathophysiology of nerve injury.     

Somatotopic redistribution of c-fos expressing neurons in the superficial dorsal horn after peripheral nerve injury

The functional somatotopic reorganization of the lumbar spinal cord dorsal horn after nerve injury was studied in the rat by mapping the stimulus-evoked distribution of neurons expressing proto-oncogene c-fos. In three different nerve injury paradigms, the saphenous nerve was electrically stimulated at C-fibre strength at survival times ranging from 40 h to more than six months: 1) Saphenous nerve stimulation from three weeks onwards after ipsilateral sciatic nerve transection resulted in an increase in the number of Fos-immunoreactive neurons within the dorsal horn saphenous territory in laminae I-II, and an expansion of the saphenous territory into the denervated sciatic territory until 14 weeks postinjury. 2) Saphenous nerve stimulation from five days onwards after ipsilateral sciatic nerve section combined with saphenous nerve crush resulted in an increase in the number of Fos-immunoreactive neurons within the dorsal horn saphenous nerve territory, and an expansion of the saphenous nerve territory into the denervated sciatic nerve territory. 3) Stimulation of the crushed nerve (without previous adjacent nerve section) at five days, but not at eight months resulted in a temporary increase in the number of Fos-immunoreactive neurons within the territory of the injured nerve, and no change in area at either survival time. The results indicate that nerve injury results in an increased capacity of afferents in an adjacent uninjured, or regenerating nerve, to excite neurons both in its own and in the territory of the permanently injured nerve in the dorsal horn. The onset and duration of the increased postsynaptic excitability and expansion depends on the types of nerve injuries involved. These findings indicate the complexity of the central changes that follows in nerve injuries that contain a mixture of uninjured, regenerating and permanently destroyed afferents.     

Electrophysiological studies on rat dorsal root ganglion neurons after peripheral axotomy: changes in responses to neuropeptides

The effect of three peptides, galanin, sulfated cholecystokinin octapeptide, and neurotensin (NT), was studied on acutely extirpated rat dorsal root ganglia (DRGs) in vitro with intracellular recording techniques. Both normal and peripherally axotomized DRGs were analyzed, and recordings were made from C-type (small) and A-type (large) neurons. Galanin and sulfated cholecystokinin octapeptide, with one exception, had no effect on normal C- and A-type neurons but caused an inward current in both types of neurons after sciatic nerve cut. In normal rats, NT caused an outward current in C-type neurons and an inward current in A-type neurons. After sciatic nerve cut, NT only caused an inward current in both C- and A-type neurons. These results suggest that (i) normal DRG neurons express receptors on their soma for some but not all peptides studied, (ii) C- and A-type neurons can have different types of receptors, and (iii) peripheral nerve injury can change the receptor phenotype of both C- and A-type neurons and may have differential effects on these neuron types.     

Heat shock protein 27: developmental regulation and expression after peripheral nerve injury

The heat shock protein (HSP) 27 is constitutively expressed at low levels in medium-sized lumbar dorsal root ganglion (DRG) cells in adult rats. Transection of the sciatic nerve results in a ninefold upregulation of HSP27 mRNA and protein in axotomized neurons in the ipsilateral DRG at 48 hr, without equivalent changes in the mRNAs encoding HSP56, HSP60, HSP70, and HSP90. Dorsal rhizotomy, injuring the central axon of the DRG neuron, does not upregulate HSP27 mRNA levels. After peripheral axotomy, HSP27 mRNA and protein are present in small, medium, and large DRG neurons, and HSP27 protein is transported anterogradely, accumulating in the dorsal horn and dorsal columns of the spinal cord, where it persists for several months. Axotomized motor neurons also upregulate HSP27. Only a minority of cultured adult DRG neurons are HSP27-immunoreactive soon after dissociation, but all express HSP27 after 24 hr in culture with prominent label throughout the neuron, including the growth cone. HSP27 differs from most axonal injury-regulated and growth-associated genes, which are typically present at high levels in early development and downregulated on innervation of their targets, in that its mRNA is first detectable in the DRG late in development and only approaches adult levels by postnatal day 21. In non-neuronal cells, HSP27 has been shown to be involved both in actin filament dynamics and in protection against necrotic and apoptotic cell death. Therefore, its upregulation after adult peripheral nerve injury may both promote survival of the injured neurons and contribute to alterations in the cytoskeleton associated with axonal growth.     

Small sensory neurons in the rat dorsal root ganglia express functional NK-1 tachykinin receptor

The tachykinins substance P (SP) and neurokinin A, released by the C-type primary afferent fibre terminals of the small dorsal root ganglion (DRG) neurons, play important roles in spinal nociception. By means of non-radioactive in situ hybridization and whole-cell recording, we showed that the small rat DRG neurons also express the NK-1 tachykinin receptor. In situ hybridization demonstrated that the positive neurons in rat DRG sections were mainly small cells (85.9%) with diameters less than 25 microm. The remaining positive neurons (14.1%) were cells with medium diameters between 26 and 40 microm. No positive large neurons (diameters > 40 microm) were observed. Expression in small DRG neurons (diameter < 21 microm) was confirmed by in situ hybridization of isolated cells, which were demonstrated to express NK-1 receptor mRNA at a very high frequency (> 90% of small DRG neurons) and therefore were subjected to whole-cell recording. In 57 of 61 cells recorded, SP or the selective NK-1 receptor agonist [Sar9, Met(O2)11]SP (Sar-SP, 1 or 2 microM) produced a delayed vibrating inward current (50-300 nA) with a long duration of 0.5-2 h. These currents were blocked by co-application of the NK-1 receptor antagonist L-668, 169 (1 microM), but were not affected by the NK-2 antagonist L-659, 877 (2 microM). Both current-clamp recording and cell-attached single-channel recording demonstrated that the long-lasting response was due to the opening of a channel with an inward current. Employment of non-Ca2+ and Ca2+ + choline solutions revealed that this channel might be a Ca2+-permeable, non-selective cation channel. The prolonged NK-1 tachykinin response exhibited extreme desensitization. This work suggests that presynaptic NK-1 autoreceptors may be present on the primary afferent terminals in the spinal cord, where they could contribute to the chronic pain and hyperalgesia.